Bottom Line:
A reactivation procedure has been optimized and used to quantify GSTP1-1 in saliva of 30 healthy subjects with results of 42±4 mU/mg-protein.The present study represents a first indication that salivary GSTP1-1 may have a different and hitherto unknown function.In addition it fulfills the basis for future investigations finalized to check the salivary GSTP1-1 as a diagnostic biomarker for diseases.

ABSTRACTGlutathione transferases (GSTs) are a superfamily of detoxifying enzymes over-expressed in tumor tissues and tentatively proposed as biomarkers for localizing and monitoring injury of specific tissues. Only scarce and contradictory reports exist about the presence and the level of these enzymes in human saliva. This study shows that GSTP1-1 is the most abundant salivary GST isoenzyme, mainly coming from salivary glands. Surprisingly, its activity is completely obscured by the presence of a strong oxidizing agent in saliva that causes a fast and complete, but reversible, inactivation. Although salivary α-defensins are also able to inhibit the enzyme causing a peculiar half-site inactivation, a number of approaches (mass spectrometry, site directed mutagenesis, chromatographic and spectrophotometric data) indicated that hypothiocyanite is the main salivary inhibitor of GSTP1-1. Cys47 and Cys101, the most reactive sulfhydryls of GSTP1-1, are mainly involved in a redox interaction which leads to the formation of an intra-chain disulfide bridge. A reactivation procedure has been optimized and used to quantify GSTP1-1 in saliva of 30 healthy subjects with results of 42±4 mU/mg-protein. The present study represents a first indication that salivary GSTP1-1 may have a different and hitherto unknown function. In addition it fulfills the basis for future investigations finalized to check the salivary GSTP1-1 as a diagnostic biomarker for diseases.

pone-0112797-g001: Inactivation of Alpha, Mu and Pi class GSTs by human saliva.(A) GSTP1-1 (open circle) (20 pmoles), GSTA1-1 (open square) (20 pmoles), GSTM2-2 (open diamond) (20 pmoles) were incubated (25°C) with 70 µl of saliva. GSTP1-1 was also incubated with the same salivary sample diluted 1∶10 (full circle). (B) GSTP1-1 (open circle) inactivated as in A for 20 min and then treated with 1 mM DTT at 37°C. Each experiment was performed in triplicate (i.e. three different spectrophotometric determinations on the same salivary sample). Error bars represent SEM.

Mentions:
Under the usual assay conditions for GST activity (1 mM GSH and 1 mM CDNB in 0.1 M potassium-phosphate buffer, pH 6.5) no detectable catalysis could be observed in 20 different saliva samples obtained from healthy subjects as described under Experimental Procedures. To check the possible presence of salivary GST inhibitors, we performed various controls. Saliva samples were supplemented with variable amounts of GSTP1-1, GSTM2-2 or GSTA1-1, as representative GSTs belonging to the most abundant classes expressed in humans (Alpha, Pi and Mu) [6]. While the activity of GSTA1-1 and GSTM2-2 in the samples was satisfactory (95–100%), the one of GSTP1-1 was less than 1% (Figure 1A). The observed inactivation was reversible and likely due to an oxidative event. In fact, treatment of these samples with 1 mM DTT at room temperature for 120 min restored the expected activity (Figure 1B). Really, exogenous GSTP1-1 underwent a fast inactivation in saliva (Figure 1A) and even diluted saliva samples were still able to inactivate this enzyme in a few minutes (Figure 2). Saliva samples not supplemented with exogenous GSTP1-1, and treated with DTT, also disclosed a detectable GST activity ranging from 30 to 50 mU per mg of salivary proteins. The observed fast inactivation is not a catalytic event but likely caused by a stoichiometric interaction of the enzyme with a component of the saliva. In fact, fixed amounts of saliva incubated with variable over-stoichiometric levels of GSTP1-1 gave the same amount of inhibited GSTP1-1 (not shown). Furthermore, incubation of a fixed amount of the enzyme with saliva samples at increasing dilutions gave different levels of inhibited enzyme correlated to the different dilutions (Figure 2). From these data and assuming a 1∶1 stoichiometry for the reaction GST-inhibitor, we estimated the concentration of the inhibitor from 5 to 15 µM in ten different saliva samples.

pone-0112797-g001: Inactivation of Alpha, Mu and Pi class GSTs by human saliva.(A) GSTP1-1 (open circle) (20 pmoles), GSTA1-1 (open square) (20 pmoles), GSTM2-2 (open diamond) (20 pmoles) were incubated (25°C) with 70 µl of saliva. GSTP1-1 was also incubated with the same salivary sample diluted 1∶10 (full circle). (B) GSTP1-1 (open circle) inactivated as in A for 20 min and then treated with 1 mM DTT at 37°C. Each experiment was performed in triplicate (i.e. three different spectrophotometric determinations on the same salivary sample). Error bars represent SEM.

Mentions:
Under the usual assay conditions for GST activity (1 mM GSH and 1 mM CDNB in 0.1 M potassium-phosphate buffer, pH 6.5) no detectable catalysis could be observed in 20 different saliva samples obtained from healthy subjects as described under Experimental Procedures. To check the possible presence of salivary GST inhibitors, we performed various controls. Saliva samples were supplemented with variable amounts of GSTP1-1, GSTM2-2 or GSTA1-1, as representative GSTs belonging to the most abundant classes expressed in humans (Alpha, Pi and Mu) [6]. While the activity of GSTA1-1 and GSTM2-2 in the samples was satisfactory (95–100%), the one of GSTP1-1 was less than 1% (Figure 1A). The observed inactivation was reversible and likely due to an oxidative event. In fact, treatment of these samples with 1 mM DTT at room temperature for 120 min restored the expected activity (Figure 1B). Really, exogenous GSTP1-1 underwent a fast inactivation in saliva (Figure 1A) and even diluted saliva samples were still able to inactivate this enzyme in a few minutes (Figure 2). Saliva samples not supplemented with exogenous GSTP1-1, and treated with DTT, also disclosed a detectable GST activity ranging from 30 to 50 mU per mg of salivary proteins. The observed fast inactivation is not a catalytic event but likely caused by a stoichiometric interaction of the enzyme with a component of the saliva. In fact, fixed amounts of saliva incubated with variable over-stoichiometric levels of GSTP1-1 gave the same amount of inhibited GSTP1-1 (not shown). Furthermore, incubation of a fixed amount of the enzyme with saliva samples at increasing dilutions gave different levels of inhibited enzyme correlated to the different dilutions (Figure 2). From these data and assuming a 1∶1 stoichiometry for the reaction GST-inhibitor, we estimated the concentration of the inhibitor from 5 to 15 µM in ten different saliva samples.

Bottom Line:
A reactivation procedure has been optimized and used to quantify GSTP1-1 in saliva of 30 healthy subjects with results of 42±4 mU/mg-protein.The present study represents a first indication that salivary GSTP1-1 may have a different and hitherto unknown function.In addition it fulfills the basis for future investigations finalized to check the salivary GSTP1-1 as a diagnostic biomarker for diseases.

ABSTRACTGlutathione transferases (GSTs) are a superfamily of detoxifying enzymes over-expressed in tumor tissues and tentatively proposed as biomarkers for localizing and monitoring injury of specific tissues. Only scarce and contradictory reports exist about the presence and the level of these enzymes in human saliva. This study shows that GSTP1-1 is the most abundant salivary GST isoenzyme, mainly coming from salivary glands. Surprisingly, its activity is completely obscured by the presence of a strong oxidizing agent in saliva that causes a fast and complete, but reversible, inactivation. Although salivary α-defensins are also able to inhibit the enzyme causing a peculiar half-site inactivation, a number of approaches (mass spectrometry, site directed mutagenesis, chromatographic and spectrophotometric data) indicated that hypothiocyanite is the main salivary inhibitor of GSTP1-1. Cys47 and Cys101, the most reactive sulfhydryls of GSTP1-1, are mainly involved in a redox interaction which leads to the formation of an intra-chain disulfide bridge. A reactivation procedure has been optimized and used to quantify GSTP1-1 in saliva of 30 healthy subjects with results of 42±4 mU/mg-protein. The present study represents a first indication that salivary GSTP1-1 may have a different and hitherto unknown function. In addition it fulfills the basis for future investigations finalized to check the salivary GSTP1-1 as a diagnostic biomarker for diseases.